The microarray is a popular and effective tool in molecular biology, such as gene expression analysis, genome analysis and drug discovery. Microarrays are well known in fields where detection of a specific material such as a DNA or RNA sequence of interest is important, and methods for making and using microarrays are widely known and practiced. A microarray can be read or visualized in numerous ways to detect targets in the sample. One of the most convenient and selective methods for visualizing a microarray is via fluorescence, since these targets of interest frequently have low background levels of fluorescence, and since this detection method adapts well to automation.
Microarray methods make it possible to simultaneously analyze the relative amount of different targets in the sample based on the intensity of the fluorescence signals representing the relative abundance of targets (e.g., nucleic acid and protein). Typical applications include expression profiling, using cDNA microarrays. (Duggan, et al., Nature Genetics Supplement, 21: 10-14, 1999; Yang, et al., Nature Review Genetics, 3: 579-588, 2002) The probe immobilized on the microarray is commonly a known structure and is used to query the targets for the presence and the amount of a feature that is known to bind to the immobilized probe. This is done by contacting the microarray with a sample believed or suspected to contain a fluorescence-labeled target known to have a strong binding affinity for the probe. Then, if the target is present in the sample, it binds to the special probe immobilized on the chip or slide and remains affixed to the microarray. It is possible to detect a large number of targets and to sort out the relative amount of these targets in the sample simultaneously, based on the fluorescence intensities on the microarray chip.
Two kinds of procedures, one-color approach and two-color approach, can be used when planning a microarray analysis. In a one-color procedure, a single fluorescence label is used: a labeled target that is specific for the probe of interest is hybridized or bound to each microarray. In a two-color procedure, two different fluorescence labels (e.g., Cy3 and Cy5 dyes) are used to label the targets in two samples (e.g., the treatment and the control), respectively. Where only one label is used, its intensity is expected to correlate with the amount of targets present in one sample. Where two different labels are used, the relative abundance of the targets from two samples can be simultaneously determined, provided the two fluorophores do not interact with each other at all.
In a two-color procedure for microarray analyses, a microarray is manufactured by spotting probes (e.g., cDNA fragments, oligonucleotides, proteins or tissues, etc.) onto a plate, chip or slide. Commonly, for example, a slide or plate of a size suitable for use in automated sample handling systems or suitable for use in commercial fluorescence scanners will be treated to provide a ‘sticky’ surface. Typical examples of such treated surfaces include, for example, a poly-lysine coated plate or slide, or an aminosilane coated plate or slide, each of which is suitable for immobilizing probes. Each of two samples (e.g., treatment and control, or positive selection and negative selection targets) is labeled with one distinct fluorophore (e.g., Cy5 for the treatment and Cy3 for the control). The mixture of two fluorophore-labeled samples is reacted with a microarray having a well-defined pattern of probes affixed to its surface and available for binding. Then this chip is washed to remove unbound fluorescent-labeled targets in two samples, and fluorescent images of the microarray are acquired with two channels of a fluorescence scanner. By analyzing the scanned images, the amount or ratio (e.g., Cy5/Cy3) of the two fluorescent labels at each spot on the microarray can be calculated and normalized, and background corrections can be made if necessary. Data interpretation is performed to obtain quantitative information about a variety of biological facts, which should insure that the results attain good levels of confidence.
In two-color systems, however, the two fluorescent labels may not be entirely independent of each other. When there is any spectral overlap between these two fluorophores, and the same targets present in the two samples (only with different fluorescence labels) can be bound with the probes in the same spot area on the microarray chip, it is possible for non-radiative processes such as FRET (fluorescence resonance energy transfer or Förster resonance energy transfer) to occur. FRET is distance sensitive, so it is not problematic unless the two different fluorophores are in close proximity; however, it is no always possible to know the distance between the two fluorophores. In addition, if either of the two fluorescent labels is affected by the irradiation used to induce fluorescence in the other (the excitation energy), or by the fluorescence emission of the other there can also be radiative errors referred to as cross-talk. This effect could be caused by direct effects of one fluorophore on the other (e.g., emission of one label affects fluorescence of the other) or instrumental (e.g., fluorescence emissions from one label are detected in the detection channel intended for the other label.) As a result of either of these, fluorescence detection in a two-color system (using two different fluorophores having different colors by virtue of their different characteristic emission wavelengths) will be potentially distorted by FRET among different fluorophore-labeled targets and/or cross-talk.
FRET is a non-radiative process in which energy is transferred from a donor fluorophore to an acceptor fluorophore when the spectral overlap of the donor emission wavelength and the acceptor excitation wavelength exists and the two different fluorophores are near each other, typically within the 1-10 nm range. The donor fluorophore (e.g., Cy3) and the acceptor fluorophore (e.g., Cy5) are a FRET fluorophore pair. The donor will be the higher-energy/shorter wavelength fluorophore; the acceptor will be the lower energy/longer wavelength fluorophore. To the extent a FRET effect occurs, the signal observed from the donor will not accurately represent the amount of donor present: FRET will reduce the amount of observable radiative emission from the donor and distort the observed fluorescence quantities or ratios. It may also affect the apparent fluorescence yield from the acceptor, since it provides an additional source of excitation in addition to the excitation energy provided by the instrument used to observe the acceptor when donor and acceptor fluorophores are excited simultaneously by two corresponding lights.
To eliminate this distortion, appropriate fluorophores should be selected to avoid interactions, and the distance between fluorophores in the sample should be beyond the range where FRET effects are strong, i.e., the fluorophores should generally be at least about 10 nm apart. Of course, in some systems it is not possible to control the distance between the fluorophores, and certain experiments may actually cause two different fluorescent labels to be held near one another.
Cross-talk is often the result of spectral bands, either emission or absorption bands, that are wide enough to permit leakage between the excitation and/or detection of the donor fluorophore and the excitation and/or detection of the acceptor fluorophore. When scanning the two-color microarray with the donor channel of the fluorescence scanner, cross-talk is exemplified by an emission from an acceptor fluorophore that arises from excitation intended for a donor fluorophore and enters the channel for donor emission detection: if the excitation energy intended for the donor fluorophore has any spectral overlap with the absorption spectrum of the acceptor fluorophore, there is potential for fluorescence from the acceptor that would not have been seen in the typical situation where its fluorescence is solely attributable to the excitation energy intended for the acceptor. It can be the reverse when scanning the two-color microarray with the acceptor channel of the fluorescence scanner: emission from a donor that arises due to, excitation intended for the acceptor and enters the channel for acceptor emission detection.
In an ideal microarray system (including a particular combination of fluorophores, and using a fluorescence scanner), both FRET and cross-talk would be avoided, so the measured fluorescence intensities would be directly proportional to the amount of each label present in each spot of the microarray.
However, the emission and excitation spectra of most members of the fluorophores used in a two-color microarray experiment have at least some overlap, and the distance between the fluorophores can hardly be controlled, especially when the fluorescent labels are used to observe two molecules that are intended to be in close association. Thus the possible effects of FRET and/or cross-talk cannot be entirely avoided for the two-color microarray experiment where dual fluorescent labels are desirable. In order for the measurements to be meaningful in a two-color microarray experiment where FRET occurs, the donor emission should be corrected for determining the quantity of donor fluorophore present. In the previous microarray experiment, fluorescence intensities of two fluorophores are acquired through scanning the microarray chip with two channels (usually Cy3 channel and Cy5 channel) of a fluorescence scanner directly and are not corrected for the distortion of FRET and/or cross-talk.
As used herein, where two fluorophores (e.g., Cy3 and Cy5) are present together in the same spot area on the chip, the higher-energy emitter (e.g., Cy3) of the two fluorophores will be referred to as the donor fluorophore. This is because the higher-energy emitter can provide enough energy to excite the lower-energy emitter, while the lower energy emitter cannot provide sufficient energy to cause the higher-energy emitter to fluoresce.
Where it occurs, a FRET interaction between donor fluorophore (e.g., Cy3) and acceptor fluorophore (e.g., Cy5) in the microarray spot will lead to a decrease in the detected emission from the donor, because the energy of the donor can transfer energy by the non-radiative FRET pathway to the acceptor, instead of emitting a detectable photon via the desired radiative relaxation pathway. Since only photons emitted via the radiative pathway are detected by the donor channel of the fluorescence scanner (the detector that looks for photons of the wavelength that is characteristic of the donor), energy lost from the donor fluorophore via the non-radiative (FRET) pathway will not be detected, and the observed signal will under-represent the amount of donor present in the spot of the microarray.
The present invention provides a reliable measurement of the fluorescence intensities of both fluorophores in a two-color microarray experiment using a three-channel fluorescence scanner. Each channel of the scanner observes a particular emission wavelength that is associated with a particular excitation wavelength, and the combination of three channels provides sufficient information to correct the measured fluorescence intensities from the microarray for the effects of FRET and/or cross-talk. The reliable fluorescence measurement method in the microarray experiment can provide accurate intensities of two fluorophores and can be used for the accurate data analysis and reliable data interpretation in a two-color microarray analysis, by providing simple methods to correct for any distortion due to FRET and/or cross-talk.
(Donor channel refers to detection where the excitation wavelength and detected emission wavelength correspond to donor. Acceptor channel refers to detection where the excitation wavelength and detected emission wavelength correspond to those of the acceptor. FRET channel refers to detection where the excitation wavelength is selected according to the donor, and detection wavelength is selected according to the acceptor emission wavelength.)